The assembly of herpesviruses is complex and includes both nuclear and cytoplasmic sites of assembly. Studies of the assembly of a-herpesviruses have lead to a model of nuclear capsid formation, envelopment/development at the nuclear membrane, and secondary envelopment of the tegumented particle in the cytoplasm in proximity of the trans-Golgi network. In contrast, the assembly of the (3-herpesviruses, human cytomegalovirus (HCMV), is less well understood and appears to differ from that of a-herpesviruses in several respects. The tegument of HCMV is acquired in both the nucleus and cytoplasm and a nuclear envelope/de-envelopment event has not been observed during HCMV assembly. In addition, the expression of several essential HCMV tegument proteins is limited to the cytoplasm of infected cells suggesting that the final envelopment of the particle could be linked to cytoplasmic tegumentation. We have proposed that envelopment and assembly of HCMV requires specific interactions between tegument and envelope glycoproteins that lead to assembly of infectious particle in the cytoplasmic assembly compartment. Previous studies have demonstrated that the tegument protein, pp28, is essential for assembly of infectious virus and deletion of this viral protein results in the accumulation of non-enveloped, tegumented capsids in the cytoplasm. Our studies have shown that localization of pp28 to the site of virus envelopment requires viral functions and that interaction with HCMV envelope glycoproteins can relocalize pp28 within the secretory pathway. Furthermore, our studies have demonstrated that pp28 multimerizes and, that posttranslational modification might contribute to its intracellular localization. In this proposal we will identify and characterize structural domains on the cytoplasmic tails of two envelope glycoproteins and in pp28 that direct the intracellular localization and virion incorporation of these virion proteins. In addition, we will define interactions between pp28 and envelope glycoproteins and determine the role of these interactions in intracellular trafficking and virion incorporation of these proteins. Finally, we propose to identify additional viral and cellular proteins that interact with pp28 and envelope proteins during assembly and to explore the role of a cellular pathway that is thought to play a role in virus budding. We believe these studies will provide a greater understanding of the process of cytoplasmic envelopment of HCMV and perhaps, identify new targets and strategies for antiviral drug development.